Negative-strand RNA viruses are associated with many diseases in humans such as influenza, rabies or measles. Other well known examples are the viral infections caused by Mumps virus, Respiratory Syncytial virus, Human Parainfluenza virus types 1-4, Ebola virus, Marburg virus, Hanta virus, Nipah virus, Vesicular Stomatitis virus, Rinderpest virus and canine Distemper virus (2). There are still many diseases associated with negative-strand RNA viruses, such as Parainfluenza virus, responsible for 30-40% of all acute respiratory infections in children and infants, for which no effective drugs or vaccines exist. Some of these viruses may re-emerge from animal species or reappear as new agents of bioterrorism. Moreover, measles virus still remains one of the leading causes of death by infectious agents worldwide. This, together with the insufficient therapy options today, has increased markedly the demand for new antiviral strategies.
Among negative-strand RNA viruses, the non-segmented negative-strand RNA viruses (Mononegavirales) are enveloped viruses that have genomes consisting of a single RNA molecule of negative sense. This order includes viruses with high medical relevance, such as the Rhabdoviridae, Paramyxoviridae, Filoviridae, and Bomaviridae families which are considered for the purpose of the invention. Although these viruses have distinct biological properties, their replicative and transcriptional system is conserved. Accordingly, the description of the invention which follows which is provided by reference to particular examples of viruses in this order, should be understood as providing disclosure of the corresponding features for other viruses of the order of Mononegavirales unless technically irrelevant for the skilled person.
The use of mammalian cells and reverse genetic tools to study negative-strand RNA virus has constituted a major advance for the comprehension of the biology of this group of pathogens and for the generation of vaccines (3). While positive-strand RNA or DNA viruses can be easily obtained in vitro after transfection of their engineered infectious cDNA or DNA in appropriate cells, the negative-strand RNA viruses cannot be rescued directly by reverse genetics from their cDNA. The genome of negative-strand RNA viruses is not able to initiate in vitro an infectious cycle because it does not code directly for proteins. Both transcription and replication require a transcriptase-polymerase enzymatic complex contained in the nucleoproteins encapsidating the viral genome (RNPs). Thus, the generation of recombinant negative-strand RNA viruses from cDNA involves reconstitution of active RNPs from individual components: RNA and proteins, to assemble nucleocapsids.
A remarkable set of work from numerous laboratories has allowed the establishment of different systems for rescuing almost all negative-strand RNA viruses from their cDNA (3). In contrast to the viruses with segmented genomes, the RNPs of non-segmented negative-strand RNA viruses (Mononegavirales) are tightly structured and contain, in addition to the nucleoprotein (N), the assembly and polymerase cofactor phosphoprotein (P) and the viral RNA polymerase large protein (L). The first infectious Mononegavirales, the rabies rhabdovirus, was recovered from cDNA in 1994 (4). The approach involved intracellular expression of rabies virus N, P, and L protein, along with a full length RNA whose correct 3′ end was generated by the hepatitis delta virus (HDV) ribozyme. A transcript corresponding to the viral antigenome (positive strand) rather than to genome (negative strand) was used to avoid a severe antisense problem raised by the presence of N, P, and L sequences in full-length RNAs. In this system, the essential helper proteins were provided by a replication-competent vaccinia vector encoding the phage T7 RNA polymerase to drive T7-specific transcription of plasmids encoding the required proteins N, P and L. Similar systems allowed recovery of infectious rabies viruses, VSV, as well as the Paramyxoviridae Sendai virus, HPIV-3 and measles virus (3).
However, in previously described methods for generating negative-strand RNA viruses by reverse genetics from infectious cDNA it is often relied on transformed mammalian cell lines that would be inappropriate for GMP (good manufacturing production) production of clinical vaccine lots, according to the certification of international safety agencies. The development of an alternative reverse genetics system for Mononegavirales in yeast would therefore be extremely advantageous. Production in yeast has especially many advantages on the industrial scale.
In order to provide an alternative to the use of mammalian cells, the inventors have considered yeast strains, especially Saccharomyces strains.
The straightforward genetics of the budding yeast Saccharomyces cerevisiae and its high conservation of basic cellular processes with higher organisms make it an excellent tool for fundamental research and drug development (5). Yeast is frequently used to produce vaccines based on recombinant proteins or virus-like-particles. For example, the efficient and safe prophylactic HPV vaccine GARADASIL® is composed of recombinant HPV VLPs antigens that are produced in yeast (6). The advantages of yeast-based vaccines are the ease of manipulation and cultivation of S. cerevisiae and the use of the fermentation process to provide large amounts of viral particles. The budding yeast is a eukaryotic organism that can be also used as a simpler system to replicate live mammalian viruses and thus, to provide substrates to produce viral live-attenuated vaccines (7). Indeed, yeast has been used successfully as a model host to replicate a wide range of viruses. These include DNA and RNA viruses that infect plants, mammals and humans (8) (9) (10) (11). However, there is not yet such technology for negative-strand RNA viruses.
Viruses that replicate in yeast comprise two families of viruses: (i) DNA viruses including dsDNA (Human papillomavirus (11), Bovine papillomavirus (12) and ssDNA (Mung bean yellow mosaic India virus) and (ii) positive strand RNA viruses family including Brome mosaic virus (8), Carnation Italian ringspot virus (13), Tomato bushy stunt virus (9), Flock House virus (10) and Nodamura virus (14). Viruses that replicate in yeast have positive-strand RNA genomes and share a common replication process: the genomic positive-strand RNA genome serves as mRNA and as template for replication. This feature facilitates the replication and the transcription of this RNA virus family in yeast. Experimentally, the strategies used to replicate positive-strand RNA virus in budding yeast have some common traits. The viral RNA-dependent RNA polymerase and viral replication essential cofactors, if required, are expressed from yeast promoters. Next, positive-strand RNA genome is introduced into yeast cells either by spheroplast transformation or by in vivo transcription from a yeast expression vector. The integrity of the 5′ and 3′ ends of the RNA is respected because they harbor important replication elements. This can be achieved by using a ribozyme to generate the exact ends. The genomic RNA contains a reporter gene, which expression is dependent on viral replication system. Stable expression of all the components of the replicative system is achieved by using yeast plasmids carrying selectable markers. The expression of the reporter gene, which depends on viral RNA replication, indicates the presence of RNA virus replication (7).
Yeast technology and the so-called <<humanized yeast>> systems have a high impact in the understanding of the host/virus-related molecular process and are potential tools to discover novel medicinal compounds (7, 15). Many studies using genome-wide screening, DNA and protein micro arrays, deletion mutants libraries, expression profiling, genome wide synthetic lethal screens and gene dosage effects have allowed the identification in yeast of multiple host factors that affect positive-strand RNA/DNA replication and are involved in unexpected novel cellular pathways (16).